Clostridium difficile initiates disease in the gastrointestinal tract and causes extracolonic damage by releasing toxins (TcdA and TcdB) into the bloodstream. Unfortunately, while much is known about the localized effects of these toxins at the site of infection, our understanding of the systemic effects of these toxins is limited. Recently, we reported that TcdB is a potent cardiotoxin, which alters cardiac function and damages the heart. However, cardiac damage could be completely alleviated by a caspase-3 inhibitor, in contrast to in vitro studies, which show only partial protection against TcdB using this inhibitor. These results suggest in vivo apoptotic cell death differs from that studied in vitro. The first series of experiments will elucidate the steps in apoptosis as they occur in vivo, in order to gain relevant insight into the activities of this toxin within the host. To date, almost nothing is known about the systemic effects of TcdA, although like TcdB, TcdA is a potent exotoxin. Using experimental approaches similar to those employed for TcdB, in the second aim of this study, we will elucidate the systemic effects of TcdA and determine this toxin may work in combination with TcdB. Finally, C. difficile associated disease is difficult to treat due to a high level of antibiotic resistant strains. In the final aim of this study we will explore several novel therapeutics designed to prevent systemic damage by TcdB and TcdA. The specific aims of this study are as follows: Specific Aim 1: The mechanism of TcdB-induced apoptosis in cardiac tissue will be determined. Specific Aim 2: The contribution of TcdA to systemic damage will be determined. Specific Aim 3: Candidate inhibitors of TcdA and TcdB will be evaluated for protection against systemic damage. Relevance: C. difficile associated disease is an increasing medical problem in many developed countries. Furthermore, there has been a concerning increase in mortality associated with this illness, yet little is known about steps in pathogenesis outside of the gastrointestinal tract. The studies proposed herein will provide important insight into systemic damage occurring in this disease, and test new candidate therapeutics.